The present invention encompasses novel synthetic peptide analogs that are antagonists to Substance P, substance P like peptides and related peptides and are useful for the treatment of cancer. The invention particularly relates to the design and synthesis of the novel substance P antagonist analogs incorporating xcex1,xcex1-dialkylated amino acids in a site specific manner. The invention encompasses methods for the generation of these peptides, compositions containing these peptides and pharmacological applications of these peptides specifically in the treatment and prevention of cancer.
Substance P is one of the main members of the tachykinin family. The tachykinins are mammalian regulatory peptides and are present in the central and peripheral nervous systems, and in gut endocrine cells. Substance P was the first gut neuropeptide to be discovered. It is a 11 residue neuropeptide of the following sequence:
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 
(SEQ ID NO: 1)
Substance P regulates gastrointestinal motility, increases blood flow in the gut, stimulates secretion of the pancreas, salivary glands, small intestines and inhibits acid secretion. In the central nervous system, tachykinins play a role in the sensory nervous pathways and in motor control. (Dockray, G. J., 1994, 401 Gut peptides: Biochemistry and Physiology, Raven Press Ltd, New York).
The role of Substance P in cancer has been well recognized particularly in small cell lung cancer. Small cell lung cancer (SCLC) cell growth is sustained by multiple autocrine and paracrine growth loops involving neuropeptides. In the search for novel antiproliferative agents for small cell lung cancer the first substance P antagonist to be studied was [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P (Antagonist A). Substance P is structurally unrelated to bombesin/GRP and has no bombesin/GRP antagonist activity; antagonist A was found to block the secretory effects of bombesin on a pancreatic preparation, diminish bombesin/GRP binding to its receptor and inhibit mitogenesis in Swiss 3T3 cells (Jensen et al., 1984, Nature, 309; 61-63; Zachary and Rozengurt, 1986, Biochem. Biophys. Res. Commun., 137;135-141). However it did not affect mitogenesis induced by polypeptide growth factors, such as platelet-derived growth factor and epidermal growth factor. Among other congeners of substance P tested for bombesin/GRP antagonism, two compounds with inhibitory activity were identified: antagonist D, [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P, and antagonist G [Arg6,D-Trp7,9, MePhe8] substance P(6-11). Antagonist D was shown to be five fold more potent than antagonist A in preventing the cellular effects of bombesin/GRP and vasopressin in mouse 3T3 cells and in inhibiting the growth of SCLC cells in serumfree medium. Overall, in Swiss 3T3 cells both antagonists were demonstrated to have in common the ability not only to inhibit the effects of bombesin/GRP but also the effects of other neuro-peptides, including vasopressin, bradykinin, endothelin, and substance P. This is also reflected by data showing that antagonist G is ten fold less potent than antagonist D in blocking bombesin/GRP mediated mitogenesis in Swiss 3T3 cells and it is almost as potent as antagonist D in inhibiting SCLC proliferation in vitro. Antagonist D inhibited proliferation of H-510 and H-69 SCLC cells in liquid culture and in semi-solid media (IC50=5 xcexcM). Colony formation stimulated by multiple neuropeptides including vasopressin and bradykinin was also blocked by Antagonist D (Seckl, M. J. et al.,1997, Can Res, 57(1):51-4). In addition, antagonist G showed inhibition of SCLC xenografts in vivo (Wolf P. J. and Rozengurt, E., 1990, Can. Res., 50(13):3968-73; Everard, M. J., et al., 1993, Eur J Cancer, 29A(10):1450-3; Br J Cancer, 1992, 65(3):388-92)). Reeve, J. G. and Bleehen, N. M. (Biochem. Biophys. Res. Commun.,1994, 199(3):1313-19) found that treatment of lung tumor cells with Antagonist D caused a concentration-dependent loss of cell viability which was accompanied by the onset of apoptosis as defined by cytological criteria and DNA fragmentation.
Short-chain SP antagonist viz.pHOPA-D-Trp-Phe-D-Trp-Leu-Leu-NH2 (Analog R) (SEQ ID NO: 2) and its analogs were studied by the Hungarian group and were found to inhibit the proliferation of H69 SCLC cells both in vitro and in xenografts in vivo in nude mice (Int. J. Cancer, 1995,60(1):82-7). In a further extension of the work, the C-terminal peptide bond was replaced by a methylene-amino (pseudopeptide) bond. Substance P analogues: D-MePhe-D-Trp-Phe-D-TrpLeu(psi)-(CH2NH)-Leu-NH2) (SEQ ID NO: 3); Analog 6 and D-MePhe-D-Trp-Phe-DTrp-Leu-MPA (SEQ ID NO: 4): Analog 7, inhibited SCLC pro-liferation more effectively than Analog R (6: IC50=2 xcexcM; 7: IC50=5 xcexcM and R: IC50=10 xcexcM). Moreover, Analog 6 inhibited the respiratory activity of SK-MES 1 epithelial type of lung carcinoma cells in proliferating but not in the quiescent state suggesting that the anti-proliferative effect of these compounds is not due to simple cytotoxicity and these short chain SP analogues may be promising candidates as therapeutic agents in the treatment of SCLC (Nyeki, O. ,et.al., 1998, J. Pept. Sci, 4(8): 486-95). Antagonist D and its role in cancer has been described in the U.S. Pat. No. 5,434,132 and WO 88/07551.
The present invention describes peptide analogs of substance P, the preparation, and use of peptide analogs of substance P especially the Antagonist D of substance P using constrained amino acids and their use thereof for cancer therapy, alone, or in combination or as an adjunct to cancer chemotherapy.
The design of conformationally constrained bioactive peptide derivatives has been one of the widely used approaches for the development of peptide-based therapeutic agents. Non-standard amino acids with strong conformational preferences may be used to direct the course of polypeptide chain folding, by imposing local stereochemical constraints, in de novo approaches to peptide design. The conformational characteristics of xcex1,xcex1-dialkylated amino acids have been well studied. The incorporation of these amino acids restricts the rotation of xcfx86,xcexa8 angles within the molecule, thereby stabilizing a desired peptide conformation. The prototypic member of xcex1,xcex1-dialkylated aminoacids, xcex1-amino-isobutyric acid (Aib) or (xcex1,xcex1-dimethyl glycine has been shown to induce (3-turn or helical conformation when incorporated in a peptide sequence (Prasad and Balaram, 1984, CRC Crit. Rev. Biochem.16, 307-347; Karle and Balaram, 1990 Biochemistry, 29, 6747-6756). The conformational properties of the higher homologs of xcex1,xcex1-dialkylated amino acids such as diethylglycine (Deg), di-n-propylglycine (Dpg) and di-n-butylglycine (Dbg) as well as the cyclic side chain analogs of a,a-dialkylated amino acids such as 1-aminocyclopentane carboxylic acid (Ac5c), 1-amino-cyclohexane carboxylic acid (Ac6c), as 1-aminocycloheptane carboxylic acid. (Ac7c) and as 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation (Prasad, S. et al., 1995 Bioployrners, 35, 11-20; Karle, L. L.,et al., 1995, J. Amer. Chem. Soc.,117, 9632-9637). xcex1,xcex1-Dialkylated amino acids have been used in the design of highly potent chemotactic peptide analogs (Prasad, S. et al., 1996 Int. J. Peptide Protein Res. 48, 312-318.) However, the applicants are not aware of any prior art for the synthesis of novel peptide analogs, encompassed in the present invention, particularly the synthesis of such substance P peptide analogs, containing xcex1,xcex1-dialkylated amino acids. Moreover, the use of such constrained amino acids for the design of peptides possessing anti-neoplastic activity is also unknown in any previous prior art. The present invention exploits the conformational properties of such xcex1,xcex1-dialkylated amino acids for the design of biologically active peptide derivatives of substance P with specific anticancer activity. Furthermore in the prior art it has been shown that lipophilazation of bioactive peptides improves the stability, bioavailability and ability to permeate biomembranes (Dasgupta, P et al; 1999, Pharmaceutical Res. 16, 1047-1053; Gozes, I. et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 427-432).
The present invention also includes synthesized peptide derivatives having N-terminal alkanoyl groups from 2-18 carbon atoms with improved anticancer activity.
In the formula (I) below and throughout the specification and claims the amino acids residues are designated by their standard abbreviations. Amino acids denote L-configuration unless otherwise indicated by D or DL appearing before the symbol and separated from it by hyphen.
The following abbreviations are also used in this application:
The present invention comprises polypeptides of the following general formula:
X-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-R-NH2
wherein,
X is acetyl or straight, branched, or cyclic alkanoyl group from 3 to 18 carbon atoms, or is deleted;
R is Aib, Deg, Dpg, AcSc or Ac6c; or R is a hydrolyzable carboxy protecting group; or a pharmaceutically acceptable salt of the peptide.